Lactoferrin is a member of the transferrin family of non-heme iron binding glycoproteins (Aisen et al., 1980, Ann. Rev. Biochem. 49:357-393), which includes transferrin, the major iron-transport protein in blood (MacGillivray et al., 1983, J. Biol. Chem. 258:3543-3553), ovotransferrin, an avian egg white protein (Jeltsch et al., 1982, Eur. J. Biochem. 122:291-295) and melanotransferrin, a membrane bound form of this family found in human melanocytes (Rose et al., 1986, Proc. Natl. Acad. Sci. USA 83:1261-1265). Lactoferrin has a broad distribution, present in both external secretions that bathe the body surfaces (Masson et al., 1971, Comp. Biochem. Physiol. 39: 119-129; Hennart et al., 1991, Am. J. Clin. Nutr. 53:32-39; Masson et al., 1966, Clin. Chim. Acta. 14:735-739; Pentecost et al., 1987, J. Biol. Chem. 262:10134-10139; Yu et al., 1993, Biochem. J. 296:107-111) and in the secondary granules of polymorphonuclear neutrophils where it can be released into the bloodstream upon neutrophil activation (Masson et al., 1969, J. Exp. Med. 130:643-658). The functions proposed for lactoferrin include iron binding and delivery to the small intestine (Fransson et al., 1980, J. Pediatrics 96:380-384; Iyer et al., 1993, Eur. J. Clin. Nutr. 47:232-241; Cox et al., 1979, Biochem. Biophys. Acta. 558:129-141; Hu et al., 1988, Biochem. J. 249:435-441; Gislason et al., 1995, J. Pediatr. Gastroent. Nutr. 21:37-43; Mikogami et al., 1994, Am. J. Physiol. 267:G1-G8; Ward et al., 1995, Biotechnology 13:498-503), antimicrobial activity against a wide range of gram-negative and gram-positive bacteria (Oram et al., 1968 Biochem. Biophys. Acta. 170:351-365; Arnold et al., 1977, Science 197:263-265; Ellison et al., 1988, Infect. Immun. 56:2774-2781; Bellamy et al., 1992, Biochem. Biophys. Acta. 1221:130-136; Yamauchi et al., 1993, Infect. Immun. 61:719-728), cellular growth promotion (Hashizume et al., 1983, Infect. Immun. 763:377-382; Nichols et al. 1987, Pediatr. Res. 21:563-567), regulation of myelopoiesis (Sawatzki et al., 1989, Blood Cells 15:371-375; Broxmeyer et al., 1986, Blood Cells 13:31-48; Zucali et al., 1979, Blood 54:951-954), and immunomodulatory properties (Machnicki et al., 1993, Int. J. Exp. Path. 74:433-439; Crouch et al., 1992, Blood 80:235-240; Zagulski et al., 1989, Br. J. Exp. Pathol. 70:697-704).
Lactoferrin shares a high degree of structural homology with other members of the transferrin family. All of these proteins are monomeric glycoproteins with a molecular weight of .about.80 kDa. Aisen et aL, 1980, Ann. Rev. Biochem. 49:357-393; Metz-Boutique et al., 1984, Eur. J. Biochem. 145:659-676. The three dimensional structure of lactoferrin (Anderson et al., 1989, J. Mol. Biol. 209:711-734) and transferrin (Lindley et al., 1988, Biochem. 27:5804-5812) have been precisely defined by X-ray crystallographic analysis. The proteins are folded into two globular lobes corresponding to the amino- and carboxy-terminal halves of the protein. This bilobal structure, with .about.40% conservation between the amino- and carboxy-terminal halves, is thought have evolved by intragenic duplication from a common ancestral gene. Williams et al., 1982, Trends Biochem. Sci. 7:394-397. Each of these lobes can reversibly bind iron with high affinity and with the concomitant binding of an anion, usually carbonate. Aisen et al., 1980, Ann. Rev. Biochem. 49:357-393. The amino acids required for iron binding by lactoferrin are highly conserved between members of the transferrin family. Baker et al., 1992, J. Inorg. Biochem. 47:147-160. In lactoferrin, an iron atom binds to Asp 396, Tyr 93, Tyr 193 and His 254 in the amino-terminal lobe, and the corresponding Asp 396, Tyr 436, Tyr 539 and His 598 in the carboxy-terminal lobe, respectively. Anderson et al., 1989, J. Mol. Biol. 209:711-734.
Nonwithstanding their structural similarities, lactoferrin displays much more avid iron binding properties than its serum counterpart, transferrin. In particular, the release of iron from lactoferrin displays greater pH stability than transferrin, the latter releasing iron in a pH range of about six (6) to about four (4) while the former releases iron a pH range of about four (4) to about two (2). Mazuier et al., 1989, Biochem. Biophys. Acta. 629:399408. It has been suggested that the unique iron binding properties of lactoferrin contribute to some of the diverse functional activities proposed for this protein. Elucidation of the structural and functional features involved in the unique iron binding properties of lactoferrin will allow the modelling and generation of lactoferrin variants with improved properties, including, but not limited to, lactoferrin variants with higher affinity for iron for improved antimicrobial activities, lactoferrin variants with lower affinity for iron having improved iron releasing properties, or lactoferrin variants having modified pH dependent efficiencies for iron release.
The Applicants of the present invention have previously reported the high level production and characterization of recombinant human lactoferrin in the filamentous fungi, including Aspergillus. Ward et al., 1995, Biotechnology 13:498-503; U.S. Pat. Nos. 5,571,896, 5,571,619, and 5,571697, incorporated by reference in their entirety. Nonwithstanding its distinct and unique carbohydrate composition, the recombinant protein was shown to be indistinguishable from human breast milk lactoferrin with respect to its physiological activities, including iron and receptor binding and antimicrobial activity. Hence, the availability of this expression system provides now the production of lactoferrin variants in sufficient quantities to address the structure/function role of this protein, in order to generate variants with improved properties.